Identification of neuropeptides, flp-1 and flp-12 targeting neuromuscular system of rice root knot nematode (RRKN) Meloidogyne graminicola

Root-knot nematodes (RKNs), Meloidogyne spp, are found in all temperate and tropical areas, and are among the most damaging plant pathogens worldwide. M. graminincola is an economically important root parasite on upland, lowland and deepwater rice. FMRFamide-like peptides (FLPs) play significant role as neurotransmitters or neuromodulators in the nervous system and proposed as one of the important targets for the plant parasitic nematode management. Therefore, for the first time, we have cloned and characterized two neuropeptide genes (flp-1 and flp-12) from the cDNA of preparasitic second stage juveniles of M. graminicola. The flp-12 contains putative 22 residue long signal peptide at N-terminal suggesting function as an extra-cellular protein. We have found highly conserved motif LFRGR in flp-1. These two flp genes could be interesting and potential targets for functional validation to explore their utility for designing management strategies.

Neuropeptide signaling system has been proposed to be the potential target for the management of plant parasitic nematodes [4,5] due to its critical role in various parasitic activities like host recognition, migration and penetration, secretory activities, alimentation, and reproduction. FMRFamide-like peptides (FLPs) have shown to be widely expressed in the nervous system of root knot nematodes, Meloidogyne spp [6]. The largest families of neuropeptides in nematodes are the FLPs, which possess a C-terminal Arg-Phe-NH2 signature and play a central role in motor activities. There are 28 flp genes reported in Caenorhabditis elegans encoding at least 72 distinct peptides [7]. Likewise, 21 FLPs have been identified in economically important plant parasitic root knot nematode, M. incognita [8]. One or more FLPs are known to coexpress indicating that there is an interaction between them. Previous reports in C. elegans have indicated that, flp-1, 12 and 8 are co expressed and a mutation in flp-8 did not have any phenotypic aberration in C. elegans. But disruption of flp-1 and 12 resulted in various neuro muscular dysfunctions [9]. Further functions of flp genes are regulated by microRNAs (miRNAs) and flp-12 was identified as a potential target in pine wood nematode, Bursaphelenchus xylophilus [10]. Although studies on role of FLPs on behavior of plant parasitic nematodes are limited, a series of RNA interference studies of flp genes in M. incognita [6] and Globodera pallida [5] revealed aberrant behavioral phenotypes and migrational abilities. flp-12 is predicted to be crucial for the normal muscular function in G. pallida and M. incognita as it has interfered with the nematode migration in a sand column in response to the root defusates [11]. A similar effect can be envisaged in M. graminicola that will be very useful in designing an efficient management strategy. In view of this, for the first time, we have cloned and characterized flp-1 and flp-12 from the Indian isolates of M. graminicola using orthologous sequences present in M. incognita.

Nematode collection
Nematode infected roots were collected from the nematode culture pots and root galls were separated and used for hatching the 2 nd stage juveniles (J2s) of M. graminicola.

RNA Isolation, quality controls and cDNA Synthesis
Total RNA was extracted from J2s of M. graminicola using NucleoSpin total RNA Kit (Macherey-Nagel, Germany). The quality and quantity of each RNA sample was confirmed by using Nanodrop (Thermo Scientific). The RNA samples with 260/280 ratio from 1.9 to 2.1, 260/230 ratio from 2.0 to 2.5 were used for the analysis. One µg of the RNA sample was reverse transcribed to cDNA by using cDNA synthesis Kit (Superscript VILO, Invitrogen). polymerase (Sigma-Aldrich) and 1 µl cDNA. The PCR amplification consisted of initial denaturation at 94 0 C for 4 min, followed by 35 cycles of amplification, denaturation at 94 0 C for 60 s, annealing at 60 0 C for 30 s and extension at 72 0 C for 1 min with a final extension at 72 0 C for 10 min. The amplified product was separated on 1.2% Agarose gel electrophoresis to confirm the size of the amplified product.

Cloning and Sequencing
Fresh PCR product was cloned into pGEM-T easy cloning vector (Promega, USA) using standard protocol. Freshly prepared competent cells of Escherichia coli DH5α were transformed with the recombinant plasmids. Positive clones were selected by blue white colony screening along with ampicillin and colony PCR. Recombinant colonies were used for plasmid extraction. Inserts in the clones were confirmed by restriction digestion with EcoRI. The positive clones were custom sequenced by ABI SOLiD sequencing system [12].

Bioinformatics Analysis
The amino acid sequences were deduced from the corresponding nucleotide sequences by using the OrfPredictor (ORF-Predictor) server, which is designed for ORF prediction and translation of a batch of EST or cDNA sequences.    Figure 1B). The cloned genes were sequenced using ABI solid sequencing platform and sequences along with the predicted ORF obtained from ORF-predictor.

Characterization of the sequences
Sequences of the partial cDNAs of flp-1 (214 bp) and flp-12 (299 bp) obtained from M. graminicola were submitted to Genbank sequence database (Accession Nos. KC250005 and KC250006).The percentage of the GC content was 43% and 35% for flp-1 and flp-12 respectively. BLASTN was carried out to determine the homology against non-redundant Genbank database. flp-1 showed best hit with the M. incognita having 70% identities (E value 3e-26) and 17% Gaps. Whereas, database searches for the flp-12 revealed 99% identity with Heterodera avenae [18] and 95% with M. incoginta. Conceptually translated nucleotide sequences into the corresponding amino acids resulted in 70 (flp-1) and 91 (flp-12) amino acids.
The BLASTX algorithm was also used to compare the M. graminicola sequence with protein sequences in database. flp-1 showed the most significant match with (Score = 236, Expect = 1e-23) with that of M. incognita while flp-12 showed most significant (Score = 472, Expect = 6e-59) match with FMRFamide-like protein 12 of cereal cyst nematode, H.avenae. SignalP server was used to identify the signal peptide in both the FLPs. SignalP result did not produce significant hits for flp-  (Figure 3). Multiple sequence alignment result suggested the diversity of flp-1 among the homologous sequences. NH2 terminal of flp-1 seems to appear poorly conserved than COOH terminal and the LFRGR motif present in three repeats is highly conserved across the nematode species (Figure 3A). This strongly suggests that these conserved motifs could be essential in signal transduction mediated by GPCR (G-protein coupled receptor) [20,21]. Alignment of flp-12 revealed high sequence conservation only among the plant parasitic nematodes. Figure  3B clearly indicated that C-terminal FMRF signature motif was not amplified during the PCR experiment (As shown in the rectangular box). Phylogenetic analysis was carried out for both Mg-flp-1 and Mg-flp-12 respectively. Protein sequence for M. graminicola and M. incognita were clustered together but appeared as an out group. Interestingly, flp-1 of A. suum, an animal parasitic nematode was grouped with C. elegans, a free living nematode. flp-1 of Ditylenchus distructor and G. pallida appeared different from the other sequences ( Figure 4A). flp-12 of H. avenae, M. graminicola and M. incognita could be clustered together as they were highly similar whereas, G. pallida was as an out-group. The sequence from M. minor was slightly diverse from the two root knot and one cyst nematode species ( Figure  4B). The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model. Initial tree for the heuristic search were obtained automatically as follows. When the number of common sites was < 100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise BIONJ method with MCL distance matrix was used. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA5. The homologous sequences genebank accessions were given in Figure 4 were used in this analysis.

Conclusion:
Neuromuscular system of nematodes has been established as an important target for nematode control efforts and majority of the leading anthelminthic drugs act on targets within the neuromuscular system. M. graminicola is an obligate parasite of rice; despite of its high economical importance, very less genetic information is available. In this regard, we have cloned and characterized the two partial cDNA sequences of M. graminicola neuropeptide, flp-1 and flp-12 genes. flp-1 predicted to have role in neuropeptide signaling pathway and locomotory behavior and putative function of flp-12 is indicated its association with G protein coupled receptor activity. These two neuropeptides can be used for future molecular and genetic studies for confirming its practical utility for the management of M. graminicola.